Species of Neomelaniconion (Neo.) are aggressive toward humans and domestic animals. Some of these species play a role in the transmission of arbovirus. In Madagascar, separation of these arbovirus vectors was based on doubtful morphological features. Sequence variation of the ribosomal DNA internal transcribed spacer 2 from morphological Neomelaniconion species of the Neomelaniconion subgenus from Madagascar was investigated to assess the validity of morphologically defined taxa. Both methods showed two taxonomic groups: the Circumluteolus Group that includes Aedes (Neo.) belleci (Le Goff, Bousses, and Brunhes, 2007), Ae. (Neo.) nigropterum (Le Goff, Bousses, and Brunhes, 2007), and Ae. (Neo.) circumluteolus (Theobald, 1908); and the Sylvaticum Group that includes Ae. (Neo.) sylvaticum (Le Goff, Bousses,and Brunhes, 2007), Ae. (Neo.) fontenillei (Le Goff, Bousses, and Brunhes, 2007), and Ae. (Neo.) albiradius (Le Goff, Bousses, and Brunhes, 2007). Although sequence analysis failed to completely resolve each group, four distinct patterns of sequence variation supported by several fixed mutations were shown from specimens in Madagascar.